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• W2006758812 abstract "Although most Src family tyrosine kinases are modified by palmitoylation as well as myristoylation, Src itself is only myristoylated. Dual acylation is important for attachment to liquid-ordered microdomains or lipid rafts. Accordingly, Src is excluded from lipid rafts in fibroblasts. Evidence of partial genetic redundancy between Src and Fyn for brain-specific targets suggests that these two kinases may occupy overlapping subcellular locations. Neuronal Src (NSrc), an alternative isoform of Src with a 6-amino acid insert in the Src homology 3 domain, is highly expressed in neurons. We investigated whether this structural difference in NSrc allows it to associate with lipid rafts. We found that perinatal mouse brains express predominantly NSrc, which is partly (10–20%) in a lipid raft fraction from brain but not fibroblasts. The association of Src with brain lipid rafts does not depend on the NSrc insert but depends on the amino-terminal myristoylation signal. A crude lipid fraction from brain promotes NSrc entry into rafts in vitro. Moreover, lipid raft-localized NSrc is more catalytically active than NSrc from the soluble fraction, possibly because raft localization alters access to other tyrosine kinases and phosphatases. These findings suggest that NSrc may be involved in signaling from lipid rafts in mouse brain. Although most Src family tyrosine kinases are modified by palmitoylation as well as myristoylation, Src itself is only myristoylated. Dual acylation is important for attachment to liquid-ordered microdomains or lipid rafts. Accordingly, Src is excluded from lipid rafts in fibroblasts. Evidence of partial genetic redundancy between Src and Fyn for brain-specific targets suggests that these two kinases may occupy overlapping subcellular locations. Neuronal Src (NSrc), an alternative isoform of Src with a 6-amino acid insert in the Src homology 3 domain, is highly expressed in neurons. We investigated whether this structural difference in NSrc allows it to associate with lipid rafts. We found that perinatal mouse brains express predominantly NSrc, which is partly (10–20%) in a lipid raft fraction from brain but not fibroblasts. The association of Src with brain lipid rafts does not depend on the NSrc insert but depends on the amino-terminal myristoylation signal. A crude lipid fraction from brain promotes NSrc entry into rafts in vitro. Moreover, lipid raft-localized NSrc is more catalytically active than NSrc from the soluble fraction, possibly because raft localization alters access to other tyrosine kinases and phosphatases. These findings suggest that NSrc may be involved in signaling from lipid rafts in mouse brain. The Src family of nonreceptor tyrosine kinases (SFKs) 1The abbreviations used are: SFK(s), Src family of nonreceptor tyrosine kinases; cSrc, normal splice form of Src; GFP, green fluorescent protein; GST, glutathione S-transferase; HA, hemagglutinin; MAb, monoclonal antibody; NSrc, neuronal Src; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid; PMSF, phenylmethylsulfonyl fluoride; SH2 and SH3 domains, Src homology 2 and 3 domains, respectively; TBS, Tris-buffered saline.1The abbreviations used are: SFK(s), Src family of nonreceptor tyrosine kinases; cSrc, normal splice form of Src; GFP, green fluorescent protein; GST, glutathione S-transferase; HA, hemagglutinin; MAb, monoclonal antibody; NSrc, neuronal Src; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid; PMSF, phenylmethylsulfonyl fluoride; SH2 and SH3 domains, Src homology 2 and 3 domains, respectively; TBS, Tris-buffered saline. is broadly expressed and involved in many cell surface receptor-mediated signaling cascades (for review, see Refs. 1Brown M.T. Cooper J.A. Biochim. Biophys. Acta. 1996; 1287: 121-149Crossref PubMed Scopus (1072) Google Scholar and 2Erpel T. Courtneidge S.A. Curr. Opin. Cell Biol. 1995; 7: 176-182Crossref PubMed Scopus (281) Google Scholar). Elucidation of normal SFK function has been difficult because most stimuli that activate SFKs also activate non-SFK tyrosine kinases. Moreover, redundancy within the SFK family has confounded attributing a specific signaling process exclusively to one kinase. Gene disruption studies have sometimes revealed exclusive functions for specific SFKs in certain cell types, Src in osteoclasts (3Lowe C. Yoneda T. Boyce B.F. Chen H. Mundy G.R. Soriano P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4485-4489Crossref PubMed Scopus (285) Google Scholar) and Lck in T cells (4Molina T. Kishihara K. Siderovski D. van Ewijk W. Narendran A. Timms E. Wakeham A. Paige C. Hartmann K. Veillette A. Nature. 1992; 357: 161-164Crossref PubMed Scopus (886) Google Scholar) for instance, but, for the most part, deficiency of one SFK is compensated by others (5Lowell C.A. Soriano P. Genes Dev. 1996; 10: 1845-1857Crossref PubMed Scopus (251) Google Scholar). The subcellular localizations of SFKs have provided valuable clues toward understanding their functions. Src in focal adhesions plays a key role in integrin-dependent signaling events that affect cellular adhesion and motility (6Lipfert L. Haimovich B. Schaller M.D. Cobb B.S. Parsons J.T. Brugge J.S. J. Cell Biol. 1992; 119: 905-912Crossref PubMed Scopus (624) Google Scholar, 7Kaplan K.B. Bibbins K.B. Swedlow J.R. Arnaud M. Morgan D.O. Varmus H.E. EMBO J. 1994; 13: 4745-4756Crossref PubMed Scopus (220) Google Scholar, 8Cary L.A. Klinghoffer R.A. Sachsenmaier C. Cooper J.A. Mol. Cell. Biol. 2002; 22: 2427-2440Crossref PubMed Scopus (129) Google Scholar), and Lck is recruited to endosomes in CD2-activated T cells where it is involved in CD2 receptor internalization (9Marie-Cardine A. Maridonneau-Parini I. Ferrer M. Danielian S. Rothhut B. Fagard R. Dautry-Varsat A. Fischer S. J. Immunol. 1992; 148: 3879-3884PubMed Google Scholar). Localization of SFKs to various subcellular locations can be affected by protein-protein interactions involving their SH3 or SH2 domains (7Kaplan K.B. Bibbins K.B. Swedlow J.R. Arnaud M. Morgan D.O. Varmus H.E. EMBO J. 1994; 13: 4745-4756Crossref PubMed Scopus (220) Google Scholar, 10Fukui Y. O'Brien M.C. Hanafusa H. Mol. Cell. Biol. 1991; 11: 1207-1213Crossref PubMed Scopus (47) Google Scholar). However, lipid-lipid interactions involving amino-terminal acyl groups on SFKs are the primary mechanism for membrane localization of SFKs (11Sefton B.M. Trowbridge I.S. Cooper J.A. Scolnick E.M. Cell. 1982; 31: 465-474Abstract Full Text PDF PubMed Scopus (152) Google Scholar, 12Liang X. Nazarian A. Erdjument-Bromage H. Bornmann W. Tempst P. Resh M.D. J. Biol. Chem. 2001; 276: 30987-30994Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 13Koegl M. Zlatkine P. Ley S.C. Courtneidge S.A. Magee A.I. Biochem. J. 1994; 303: 749-753Crossref PubMed Scopus (139) Google Scholar, 14Magee A.I. Gutierrez L. Marshall C.J. Hancock J.F. J. Cell Sci. 1989; 11: 149-160Crossref Google Scholar), particularly localization to membrane microdomains or lipid rafts. Lipid rafts are “liquid-ordered” microdomains in cell membranes (15Simons K. Toomre D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 31-39Crossref PubMed Scopus (5058) Google Scholar, 16Brown D. London E. J. Membr. Biol. 1998; 164: 103-114Crossref PubMed Scopus (826) Google Scholar) which have been shown to exist in live cells at 37 °C (17Varma R. Mayor S. Nature. 1998; 394: 798-801Crossref PubMed Scopus (1017) Google Scholar, 18Pralle A. Keller P. Florin E.L. Simons K. Horber J.K. J. Cell Biol. 2000; 148: 997-1008Crossref PubMed Scopus (833) Google Scholar, 19Harder T. Scheiffele P. Verkade P. Simons K. J. Cell Biol. 1998; 141: 929-942Crossref PubMed Scopus (1038) Google Scholar). Enriched in cholesterol, sphingolipids, and phosphoinositides, these membrane microdomains contain proteins involved in vesicular trafficking and signal transduction. Lipid rafts often contain structural proteins such as caveolins and flotillins, but some do not (20Smart E.J. Graf G.A. McNiven M.A. Sessa W.C. Engelman J.A. Scherer P.E. Okamoto T. Lisanti M.P. Mol. Cell. Biol. 1999; 19: 7289-7304Crossref PubMed Scopus (917) Google Scholar). SFKs (12Liang X. Nazarian A. Erdjument-Bromage H. Bornmann W. Tempst P. Resh M.D. J. Biol. Chem. 2001; 276: 30987-30994Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar), receptor tyrosine kinases (platelet-derived growth factor receptor, epidermal growth factor receptor) (21Baron W. Decker L. Colognato H. Ffrench-Constant C. Curr. Biol. 2003; 13: 151-155Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 22Stehr M. Adam R.M. Khoury J. Zhuang L. Solomon K.R. Peters C.A. Freeman M.R. J. Urol. 2003; 169: 1165-1170Crossref PubMed Scopus (50) Google Scholar, 23Sun J. Nanjundan M. Pike L.J. Wiedmer T. Sims P.J. Biochemistry. 2002; 41: 6338-6345Crossref PubMed Scopus (75) Google Scholar, 24Roepstorff K. Thomsen P. Sandvig K. van Deurs B. J. Biol. Chem. 2002; 277: 18954-18960Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar), and G proteins (25Oh P. Schnitzer J.E. Mol. Biol. Cell. 2001; 12: 685-698Crossref PubMed Scopus (346) Google Scholar, 26Moffett S. Brown D.A. Linder M.E. J. Biol. Chem. 2000; 275: 2191-2198Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar) are reportedly found in rafts. It is thought that rafts function in signaling by concentrating signaling components, as seen during immunoglobulin E-triggered allergic responses in mast cells and basophils (27Field K.A. Holowka D. Baird B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9201-9205Crossref PubMed Scopus (269) Google Scholar, 28Sheets E.D. Holowka D. Baird B. Curr. Opin. Chem. Biol. 1999; 3: 95-99Crossref PubMed Scopus (118) Google Scholar). Conversely, exclusion of molecules from rafts may also regulate signal transduction. For instance, T cell receptor engagement leads to raft aggregation, thus facilitating colocalization of Lck, LAT, and T cell receptor while excluding CD45 phosphatase, hence enhancing protein-tyrosine phosphorylation (29Janes P.W. Ley S.C. Magee A.I. J. Cell Biol. 1999; 147: 447-461Crossref PubMed Scopus (693) Google Scholar). Although most SFKs are double acylated, with myristate on Gly-2 and palmitate on Cys-3, Src, Blk, and the p61 isoform of Hck are only myristoylated (13Koegl M. Zlatkine P. Ley S.C. Courtneidge S.A. Magee A.I. Biochem. J. 1994; 303: 749-753Crossref PubMed Scopus (139) Google Scholar, 30Robbins S.M. Quintrell N.A. Bishop J.M. Mol. Cell. Biol. 1995; 15: 3507-3515Crossref PubMed Scopus (227) Google Scholar, 31Shenoy-Scaria A.M. Dietzen D.J. Kwong J. Link D.C. Lublin D.M. J. Cell Biol. 1994; 126: 353-363Crossref PubMed Scopus (340) Google Scholar, 32Paige L.A. Nadler M.J. Harrison M.L. Cassady J.M. Geahlen R.L. J. Biol. Chem. 1993; 268: 8669-8674Abstract Full Text PDF PubMed Google Scholar, 33Yurchak L.K. Sefton B.M. Mol. Cell. Biol. 1995; 15: 6914-6922Crossref PubMed Scopus (81) Google Scholar). Dual acylation is important for the inclusion of SFKs such as Fyn, Lyn, Lck, and Hck p59 into lipid rafts of fibroblasts and leukocytes (30Robbins S.M. Quintrell N.A. Bishop J.M. Mol. Cell. Biol. 1995; 15: 3507-3515Crossref PubMed Scopus (227) Google Scholar, 31Shenoy-Scaria A.M. Dietzen D.J. Kwong J. Link D.C. Lublin D.M. J. Cell Biol. 1994; 126: 353-363Crossref PubMed Scopus (340) Google Scholar, 34Wolven A. Okamura H. Rosenblatt Y. Resh M.D. Mol. Biol. Cell. 1997; 8: 1159-1173Crossref PubMed Scopus (146) Google Scholar). Introducing a palmitoylation signal into wild-type Src causes relocalization to rafts (31Shenoy-Scaria A.M. Dietzen D.J. Kwong J. Link D.C. Lublin D.M. J. Cell Biol. 1994; 126: 353-363Crossref PubMed Scopus (340) Google Scholar). The exclusion of Src from rafts implies that it may not encounter substrates that are localized in or traffic through lipid rafts, and hence Src may be performing distinct functions from raft-localized SFKs. This notion is supported by the finding that EphrinA5 tyrosine phosphorylation in brain lipid rafts is reduced when Fyn is absent, even though Src is present (35Davy A. Gale N.W. Murray E.W. Klinghoffer R.A. Soriano P. Feuerstein C. Robbins S.M. Genes Dev. 1999; 13: 3125-3135Crossref PubMed Scopus (247) Google Scholar). On the other hand, some other brain proteins are phosphorylated by either Src or Fyn. For example, Src and Fyn are redundant for tyrosine phosphorylation of p190 RhoGAP in the brain (36Brouns M.R. Matheson S.F. Settleman J. Nat. Cell Biol. 2001; 3: 361-367Crossref PubMed Scopus (199) Google Scholar). And although Reelin-induced tyrosine phosphorylation of Dab1 in neurons appears to be primarily mediated by Fyn, Src can phosphorylate Dab1 when Fyn is absent (37Arnaud L. Ballif B.A. Forster E. Cooper J.A. Curr. Biol. 2003; 13: 9-17Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). This implies that both Fyn and Src can access p190 RhoGAP and Dab1. It is known that an alternative form of Src, neuronal Src (NSrc), is expressed during neuronal differentiation (38Brugge J.S. Cotton P.C. Queral A.E. Barrett J.N. Nonner D. Keane R.W. Nature. 1985; 316: 554-557Crossref PubMed Scopus (234) Google Scholar). NSrc differs from the normal splice form (called here cSrc) by 6 amino acids (RKVDVR) that are inserted into the SH3 domain because of an alternative splicing event (39Levy J.B. Dorai T. Wang L.H. Brugge J.S. Mol. Cell. Biol. 1987; 7: 4142-4145Crossref PubMed Scopus (97) Google Scholar, 40Martinez R. Mathey-Prevot B. Bernards A. Baltimore D. Science. 1987; 237: 411-415Crossref PubMed Scopus (211) Google Scholar). The activity of NSrc is 2–4-fold higher than that of cSrc (41Brugge J. Cotton P. Lustig A. Yonemoto W. Lipsich L. Coussens P. Barrett J.N. Nonner D. Keane R.W. Genes Dev. 1987; 1: 287-296Crossref PubMed Scopus (50) Google Scholar, 42Cartwright C.A. Simantov R. Kaplan P.L. Hunter T. Eckhart W. Mol. Cell. Biol. 1987; 7: 1830-1840Crossref PubMed Scopus (65) Google Scholar). It is thought that these 6 amino acids destabilize the inactive conformation of Src, thereby leading to enhanced activity (43Sicheri F. Kuriyan J. Curr. Opin. Struct. Biol. 1997; 7: 777-785Crossref PubMed Scopus (326) Google Scholar). It is also possible that the inclusion of these amino acids alters the localization of Src in cells and perhaps redistributes Src into lipid rafts. Consistent with this hypothesis, the NSrc insert is on a surface of the SH3 domain which is thought to be close to the membrane (43Sicheri F. Kuriyan J. Curr. Opin. Struct. Biol. 1997; 7: 777-785Crossref PubMed Scopus (326) Google Scholar). If NSrc is in lipid rafts, that may explain the observed partial redundancy with Fyn. Indeed, some reports place Src in lipid rafts, despite the absence of a palmitoylation signal. Some studies used “pan-Src” antibody, or antibodies against the phosphorylated activation loop tyrosine of Src, which do not distinguish Src from other SFKs (44Tansey M.G. Baloh R.H. Milbrandt J. Johnson Jr., E.M. Neuron. 2000; 25: 611-623Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, 45Lee H. Park D.S. Wang X.B. Scherer P.E. Schwartz P.E. Lisanti M.P. J. Biol. Chem. 2002; 277: 34556-34567Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). However, Src monoclonal antibodies have been used to show ligand-stimulated Src recruitment to EphrinB1-containing microdomains in neurons (46Palmer A. Zimmer M. Erdmann K.S. Eulenburg V. Porthin A. Heumann R. Deutsch U. Klein R. Mol. Cell. 2002; 9: 725-737Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar), and specific antibodies show the coexistence of Src, Fyn, Lyn, and Yes in rafts from neuroblastoma cells (47Prinetti A. Iwabuchi K. Hakomori S. J. Biol. Chem. 1999; 274: 20916-20924Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 48Kalka D. von Reitzenstein C. Kopitz J. Cantz M. Biochem. Biophys. Res. Commun. 2001; 283: 989-993Crossref PubMed Scopus (80) Google Scholar). Thus it is likely that Src itself is actually localized to rafts, at least in some tissues. We have investigated further whether Src itself is in lipid rafts and found that up to 20% of mouse brain Src is in lipid rafts. However, Src is not in lipid rafts in fibroblasts. As expected, the major form of Src in mouse brain is NSrc, but isoform differences do not explain the presence of Src in brain lipid rafts. When NSrc is expressed in fibroblasts it does not enter rafts. Localization to lipid rafts in brain requires only the amino-terminal myristoylation signal and not the SH3 region containing the NSrc insert and depends on brain lipids. Finally, to begin to understand the function of NSrc in rafts, we explored the kinase activity of NSrc in mouse brain and found that total NSrc kinase activity is dependent not only on the isoform but also on raft association. Cell Lines, Culture, and Transfection—SYF1 cells, derived from src-/- yes-/- fyn-/- mouse embryos using T antigen, were described previously (49Klinghoffer R.A. Sachsenmaier C. Cooper J.A. Soriano P. EMBO J. 1999; 18: 2459-2471Crossref PubMed Scopus (644) Google Scholar). To generate SYF1 cells reexpressing either mouse cSrc or mouse NSrc a standard retroviral infection protocol was used (50Miller A.D. Rosman G.J. BioTechniques. 1989; 7: 980-982PubMed Google Scholar). The retroviral vector pLXSH containing Src cDNA was used to generate viruses in 293T cells, followed by exposure of target SYF1 to these viruses at a low multiplicity of infection. The day after viral infection, SYF cells were selected with 0.2 mg/ml hygromycin B (Calbiochem). Src expression was verified by Western blotting. After the initial selection, stable cell lines (selected as pools, not clones) were maintained in the absence of hygromycin, and all cell lines were used at low passage number for all experiments. Fyn3 cells, SYF1 cells reexpressing Fyn cDNA, were provided by Dr. L. A. Cary. SYF1 cells, Src- or Fyn-reexpressing SYF cells, and human embryonic kidney 293T cell lines were all grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (Hyclone), 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). 293T cells were transfected with DNA:calcium phosphate coprecipitates using the HEPES-buffered calcium phosphate method. Transfection medium was replaced by fresh culture medium 24 h after transfection, and cells were lysed 48 h after transfection. For retrovirus production 4 ml of fresh medium was added after 48 h, and virus was collected between 60 to 72 h post-transfection. SYF cells were transfected using LipofectAMINE Plus (Invitrogen) in Opti-MEM (Invitrogen) using methods described by the manufacturer. Transfection medium was replaced with fresh culture medium 12 h post-transfection, and cells were lysed 48 h post-transfection. Animals—All mice used in this study were hybrid C57BL6J/129Sv. Genotyping typing was performed by PCR on tail DNA Expression Plasmids—Construction of mouse Src cDNA expression plasmids pSGT-cSrc, pSGT-NSrc, pLXSH-cSrc, and pLXSH-NSrc are described in Ref. 37Arnaud L. Ballif B.A. Forster E. Cooper J.A. Curr. Biol. 2003; 13: 9-17Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar. Hemagglutinin epitope (HA)-tagged cSrc and NSrc cDNAs were constructed by replacing the stop codon of Src cDNA in pSGT-Src with an XhoI site and subcloning the sequenced BamHI-XhoI fragment into pIRES-hrGFP-2a.1 (Stratagene). Use of plasmids expressing GST-NSrcSH3 and GST-FynSH3 fusion proteins are described in Ref. 51Brown M.T. Andrade J. Radhakrishna H. Donaldson J.G. Cooper J.A. Randazzo P.A. Mol. Cell. Biol. 1998; 18: 7038-7051Crossref PubMed Scopus (189) Google Scholar. Src NH2-terminal 7 amino acids were fused to GFP (pCMX-Src7aa-GFP) by first replacing the start codon of GFP in pCMX-GFP with a HindIII-ATG-KpnI cassette (pCMX-Δ ATG-GFP). A HindIII-KpnI adaptor encoding the first 7 amino acids of Src, MGSNKSK, was prepared by boiling the oligonucleotides 5′-AGCTTCCATGGGCAGCAACAAGAGCAAGGGTAC-3′ and 5′-CCTTGCTCTTGTTGCTGCCCATGGA-3′ and annealing them at room temperature. The double-stranded adaptor was purified from a 15% acrylamide-TBE gel after ethidium bromide staining, excising the DNA band, and eluting DNA from the gel piece by overnight incubation in water. Double-stranded adaptors encoding the G2A mutant, MASNKSK, were purified similarly. These adaptors were cloned into HindIII- and KpnI-digested pCMX-Δ ATG-GFP. All plasmids were sequenced to confirm the appropriate changes. Antibodies—Anti-Src monoclonal antibody (MAb) LA074 was produced from LP-016 mouse hybridoma cells (NCI Repository, Viromed Biosafety Laboratories, Camden, NJ) and diluted from an unpurified cell supernatant. This MAb was raised against amino acids 2–17 of v-Src. The anti-Src MAb 327 was a kind gift from Dr. J. S. Brugge (Harvard University). An anti-pan-Src antibody that recognizes Src, Yes, and Fyn, SRC2, and anti-Fyn antibody, FYN3, were purchased from Santa Cruz Biotechnology. Anti-phosphotyrosine antibody 4G10 was from Upstate Biotechnology. Anti-phospho-Src Tyr-418 (referred to as such for anti-phospho autocatalytic tyrosine) and anti-phospho-Src Tyr-529 (referred to as such for anti-phospho carboxyl-terminal tyrosine) antibody were from Biosource. Anti-HA antibody HA.11 was from Covance, anti-caveolin antibody from Transduction Laboratories, and anti-GFP antibody from Roche Applied Science. The anti-GST antibody 38.3 was described earlier (52Waskiewicz A.J. Flynn A. Proud C.G. Cooper J.A. EMBO J. 1997; 16: 1909-1920Crossref PubMed Scopus (776) Google Scholar). Horseradish peroxidase-conjugated secondary antibodies were purchased from Bio-Rad. Preparation of Lipid Rafts—Lipid rafts were isolated from cells and brain tissue primarily as described earlier (53Kawabuchi M. Satomi Y. Takao T. Shimonishi Y. Nada S. Nagai K. Tarakhovsky A. Okada M. Nature. 2000; 404: 999-1003Crossref PubMed Scopus (456) Google Scholar). Briefly, frozen brain tissue was weighed and homogenized in an ice-cold Triton X-100 lysis buffer containing 1% Triton X-100 and 5% glycerol in buffer A (50 mm Tris-HCl, pH 8.0, 10 mm MgCl2, 0.15 m NaCl, 20 mm NaF, 1 mm Na3VO4, 5 mm β-mercaptoethanol, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mm PMSF) at a ratio of 8:1 (v/w) by 5 strokes with a loose pestle followed by 10 strokes with a tight pestle of an all glass homogenizer, while keeping the entire apparatus chilled on ice. The brain lysates were collected in microcentrifuge tubes and centrifuged at 500 × g for 5 min in a cooled tabletop microcentrifuge to sediment nuclei and tissue debris. The supernatant was collected and used for preparation of lipid rafts. Cultured adherent cells, in culture dishes, were washed with cold phosphate-buffered saline (PBS) and scraped and collected in chilled PBS. The cells were centrifuged gently in a clinical centrifuge, and PBS was aspirated. The packed cell volume was estimated, and the cell pellet was gently resuspended in ice-cold Triton X-100 lysis buffer at a ratio of 8:1 (v/v). After mixing on ice for 20 min, the cell lysates were centrifuged at 500 × g for 5 min in a cooled tabletop microcentrifuge to sediment nuclei, and the supernatant was collected for preparation of lipid rafts. The Triton X-100 lysates collected as described above were mixed on ice for at least 1 h. Then, to 1 ml of the lysate, 1 ml of an 80% sucrose solution in buffer A was gradually added with continuous mixing on ice resulting in a solution containing 40% sucrose. 1.9 ml of this mixture was placed at the bottom of a 5-ml ultracentrifuge tube (Ultraclear, Beckman), and 2.5 ml of a 35% sucrose solution in buffer A and 0.7 ml of a 5% sucrose solution in buffer A were sequentially layered to form a small scale sucrose step gradient. These sucrose gradients were centrifuged in a Beckman SW 55 rotor for 16 h at 200,000 × g and at 4 °C. 0.5-ml fractions were collected from the top and analyzed by Western immunoblotting. Often, lipid raft material was collected from the interface between 5 and 35% sucrose in one 0.5-ml “raft” fraction, and the last four fractions were combined to produce a 2-ml mixture of “soluble” fraction. In general 50 μl of raft or soluble fractions was precipitated by mixing with 20 μl of 50% trichloroacetic acid and incubating on ice for 30 min followed by high speed centrifugation at 4 °C for 15 min. The trichloroacetic acid precipitates were washed with 250 μl of ice-cold acetone, air dried, and resuspended in 1× SDS-PAGE sample buffer prior to SDS-PAGE analysis. The raft fractions are thus overloaded by a factor of 4 relative to the nonraft fractions. However, the total protein in the raft fractions was less than 1% of the protein in the soluble fractions. For immunoprecipitation reactions (see below), raft fractions were solubilized by adding octyl β-d-glucopyranoside (Calbiochem) to a final concentration of 2%. In mixing experiments, lysates, recombinant proteins, and lipid preparations were mixed together in 1% Triton X-100 lysis buffer on ice for at least 2 h prior to mixing with 80% sucrose solution in buffer A. In some experiments, a Triton X-100-soluble fraction of a cell lysate was isolated by ultracentrifugation of a 1% Triton X-100 lysate in an airdriven ultracentrifuge (Beckman) at maximum air pressure and at 4 °C, thus precipitating insoluble material. Immunoprecipitation, Gel Electrophoresis, and Western Blot Analysis—Total protein levels in lysates were measured using the Bio-Rad protein assay dye, and equivalent amounts of protein were used for immunoprecipitation from Triton X-100 fractions. Because the total protein in lipid raft fractions was limitingly low for measurement, same volume equivalents of raft fraction as soluble fractions were used when comparing abundance of proteins in each fraction. Alternatively, for standardization purposes prior to immunoprecipitation kinase assays, or to compare phosphorylation status of proteins, a range of amounts of immunoprecipitates was analyzed. 1% Triton X-100 lysates, with or without added octyl β-d-glucopyranoside, were mixed with modified radioimmunoprecipitation assay buffer (150 mm NaCl, 50 mm Tris, pH 7.4, 2 mm EDTA, 1% sodium deoxycholate, 1% Nonidet P-40, 0.1% SDS, 1 mm Na3VO4, 20 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mm PMSF) to obtain a total volume of 600 μl. Immunoprecipitations were performed by first incubating the indicated primary antibodies with the cell lysates for 3 h or overnight at 4 °C. Immune complexes formed with rabbit antibodies were precipitated by incubating the reactions with protein A-immobilized Sepharose CL-4B beads (Sigma) for an additional 1 h at 4 °C. Immune complexes formed with mouse antibodies were immunoprecipitated by a similar incubation with either protein G-immobilized Sepharose 4 Fast Flow (Amersham Biosciences) or with rabbit anti-mouse immunoglobulin G and protein A-immobilized Sepharose CL-4B beads. Immunoprecipitates were washed three times with modified radioimmunoprecipitation assay buffer and were either resuspended in SDS-PAGE sample buffer for Western blotting or processed through additional washes in preparation for kinase assays. SDS-PAGE and Western blotting were performed as described previously (8Cary L.A. Klinghoffer R.A. Sachsenmaier C. Cooper J.A. Mol. Cell. Biol. 2002; 22: 2427-2440Crossref PubMed Scopus (129) Google Scholar); however, different acrylamide concentration in gels and different acrylamide:bisacrylamide ratios were used to obtain optimal separation of proteins. The different gel properties are described in the legends of each experiment. Most notable are the 8 or 9% acrylamide gels with a 20:1 acrylamide:bisacrylamide ratio which were used to separate Src or Fyn from the immunoglobulin G heavy chain in immunoprecipitates. The proteins were transferred to pure nitrocellulose filters (0.22 μm pore size), and the were filters stained with Ponceau S to confirm proper transfer and subsequently blocked with 2% bovine serum albumin in Tris-buffered saline (TBS) containing 0.05% Tween 20 for 1 h at room temperature and then incubated with indicated primary antibody for either 1 or 2 h at room temperature or overnight at 4 °C. After incubation with the indicated primary antibody, the filters were washed three times with TBS containing 0.5% Tween 20 (TBST) and then incubated with appropriate horseradish peroxidase-conjugated secondary antibodies. The filters were washed three times with TBST and once TBS, and immune complexes formed on the blot were visualized by enhanced chemiluminescence using the Western Lighting (PerkinElmer Life Sciences) chemiluminescence reagent. Immunoprecipitate Kinase Assays—Src immunoprecipitates using MAb LA074 or MAb 327 were washed three times with ice-cold radio-immunoprecipitation assay lysis buffer, once with buffer PAN (10 mm PIPES, pH 7, 100 mm NaCl, 20 μg/ml aprotinin) containing 0.5% Nonidet P-40, once with buffer PAN and once with kinase buffer (20 mm PIPES, pH 7, 4 mm MnCl2). Rabbit muscle enolase (Sigma) was denatured with 50 mm acetic acid for 5 min at 30 °C and buffered with 1 m PIPES, pH 7.0. Immunoprecipitates were incubated in kinase buffer containing 1 μm ATP and 5 μCi of [γ32P]ATP and 1.5 μg of acid-denatured enolase as a substrate, per reaction, for 5 or 10 min at room temperature, and the reaction was stopped by the addition of SDS-PAGE sample buffer. Samples were boiled, resolved by SDS-PAGE, and the gels were either blotted to nitrocellulose membranes, processed for Western blotting and autoradiography, or dried and directly visualized by autoradiography. V8 Protease Mapping—Src immunoprecipitates using MAb LA074 were washed three times with ice-cold radioimmunoprecipitation assay buffer and once with phosphatase buffer (50 mm Tris, pH 7.5, 0.1 mm EDTA, 5 mm dithiothreitol, 0.01% Brij 35, 2 mm MnCl2). The immunoprecipitates were incubated with 400 units of fresh λ phosphatase (New England Biolabs) at 30 °C" @default.
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• W2006758812 title "Lipid-dependent Recruitment of Neuronal Src to Lipid Rafts in the Brain" @default.
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